Murine Respiratory Mycoplasmosis, caused by Mycoplasma pulmonis remains a common problem in not only conventional, but also barrier-maintained rodent colonies. During the last two grant periods, we developed and extensively tested an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-M. pulmonis antibody. This assay was crucial for establishing the extent of M. pulmonis infection and gave the first indication that Mycoplasma arthritidis is also common in barrier-maintained colonies. However, there are several limitations of the ELISA antibody detection assay, including the inability to diagnose early, low dose infection when animals do not produce detectable amounts of serum antibody and the inability of ELISA to distinguish between infections with M. pulmonis and M. arthritidis. Modifications of the ELISA to overcome these shortcomings present several technical difficulties. Furthermore, several theoretical possibilities suggest that even if these were overcome, the assay still probably would prove inadequate for detection of low level infection. Recent advances in the fields of gel electrophoresis, monoclonal antibody production, and immunochemistry make development of an extremely sensitive specific antigen detection assay for M. pulmonis and M. arthritidis both feasible and practical. Thus, we propose to (i) isolate species specific membrane protein antigens from each by two-dimensional gel electrophoresis, (ii) develop and characterize a library of high affinity monoclonal antibodies to these proteins, (iii) develop antigen detection assays that will allow detection of early, low level infections with either organism, and (iv) determine the actual efficacy of the assays by extensive experimental and field tests. The studies outlined almost surely will meet the main goal of providing a means to detect early, low level infection in barrier-maintained colonies. In addition, they will yield information concerning the membrane proteins of the murine mycoplasms which will ultimately allow correlation of function and structure for these proteins and provide the necessary background for genetic studies. Other benefits will be the identification of proteins that may prove useful for subunit vaccines. Finally, knowledge gained from these studies should prove useful for similar studies with other pathogens including those of man.